The replication of (microbial and) eukaryotic genomes Flashcards
High Fidelity of DNA replication
Only about 1 mistake in every 10^10 nucleotides copied
Amazing accuracy – much higher than expected from
accuracy of complementary base-pairing
Error in DNA replication
Sometimes errors can occur
• With small changes in geometry, two hydrogen
bonds can form between G and T
• Rare tautomeric forms of DNA bases occur transiently causing incorrect pairing
Tautomeric Forms
Rare tautomeric forms of DNA bases occur transiently causing incorrect pairing
In the rare tautomeric form, C can pair with A instead of G, etc.
With small changes in geometry, two hydrogen
bonds can form between G and T
DNA ploymerase to rescue
High fidelity of DNA replication important
in initial base-pairing
• Correct nucleotide has a higher affinity
for polymerase (more energetically favorable)
DNA polymerase before covalent binding
• After nucleotide binding before covalent addition,
enzyme must undergo conformational change
where its fingers tighten around the active site
• Occurs more readily with correct base-pairing
• Allows polymerase to double check
DNA polymerase after covalent binding
• The next error-correcting reaction is exonucleolytic
proofreading
• DNA molecules with mismatched nucleotide at 3’
OH end not effective
DNA polymerase self corrects
• 3’-to-5’ proofreading exonuclease clips off
any unpaired residues at the primer
• DNA polymerase functions as a self correcting enzyme with 5’–3 ’ DNA
synthesis activity and 3’–5’ exonuclease
activity
A need for proofreading may explain the 5’ to 3’ direction of DNA chain growth
Growth in 5’-to-3’ direction allows chain to continue to
be elongated when a mistake has been removed
by exonucleolyticm proofreading
Strand-directed Mismatch Repair in prokaryotes
In E.coli, DNA methylation adds methyl groups to all A
nucleotides in the sequence GATC, but not immediately during replication
• Therefore, GATC sequences that have not yet been
methylated are in the new strands just behind the
replication fork
• Three step process
– Recognition of a mismatch
– Excision of the segment of DNA with mismatch
– Resynthesis of the excised segment using old strand as template
• Reduces number of errors made by factor of 100-1,000
Strand distinction in Eukaryotes
• In eukaryotes, mechanism for distinguishing newly
synthesized strand from parental template does not
depend on DNA methylation
• Newly synthesized lagging-strand transiently contains nicks which provides the signal that directs the mismatch proof reading system
• However, this also requires the newly synthesized DNA on the leading strand to be transiently nicked – how this occurs is uncertain
Model for strand-directed mismatch repair in eukaryotes
• MutS binds specifically to mismatched base
pair
• MutL scans nearby DNA for a nick, triggers
degradation of nicked strand all the way back
through the mismatch
Medical implications of mismatch repair
In humans seen in people who inherit one
defective copy of a mismatch repair gene
• Marked pre-disposition for certain cancers like
hereditary nonpolyposis colon cancer
• Spontaneous mutation of remaining
functional gene produces clone of somatic
cells that accumulate mutations very rapidly
DNA organisation in eukaryotes
• Chromosomes (found in the nucleus) – DNA content and complexity – DNA packaging – Inheritance • Extranuclear (extrachromosomal) DNA – Organelle genomes – Coding capacity – Inheritance
Eukaryotic chromosomes
• DNA is organised as fibre like-structures called
chromosomes
• Each chromosome consists of a single linear
DNA molecule
– No. of chromosomes varies between different
species
• Eukaryotic organisms generally diploid (two
copies of each chromosome)
– Prokaryotes are haploid (one copy per cell)
Chromosome structure: chromatin
DNA must be highly compacted to fit into the cell
